HSV ABVIC Test

Herpes Antibody Tests

In this blog post, I begin to describe the HSV-Type-Specific ABVIC Test.  Regarding the acronym ABVIC, this stands for “Antibody-Binding to Virus-Infected Cells.”   For starters, I address four key questions about the ABVIC Test which has been under development since 2011 and which is now patented and soon-to-be licensed by my company, Rational Vaccines (RVx), Inc.

  1.   How does ABVIC compare to the Current Herpes Antibody Tests?
  2.   Practical Advantage of HSV-Type-Specific ABVIC?
  3.   Why is the HSV-Type-Specific ABVIC Test Better?
  4.   What do HSV-Type-Specific Test Results Look Like?

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1.  how does ABVIC compare to CURRENT HerpeS ANTIBODY TESTs?

My laboratory is currently in the process of finalizing and writing up a manuscript for publication that will compare the resolving power of the HSV-Type-Specific ABVIC Test to the available Herpes Antibody Tests, HerpeSelect and HSV Western Blot.  I attach a 1-page poster of the results of this study here:  Autumn Immunology Conference Poster (Nov 2015)  This poster was presented at the Autumn Immunology Conference in Chicago in Nov 2015 and will be presented at the meeting of the American Association of Immunologists in Seattle in May 2016.

In this blog post, I summarize the results of the ABVIC analysis prior to its formal publication, because I think this news is too important for the potentially millions of people who have been told by a doctor or nurse over the past 20 years that they “might have HSV-2,” based on what are scientifically flimsy test results (i.e., HerpeSelect Index Values between 1 and 5).  Within the next 3 to 4 months (pending CLIA certificiation), Rational Vaccines (RVx), Inc will be offering the HSV-Type-Specific ABVIC Test to patients whom have received past herpes antibody results that suggest they are infected with HSV-2 despite an absence of clinical signs or behavioral risk factors, then you may wish to consider RVx’s HSV-Type-Specific ABVIC Test (cost $250).  RVx’s website will be live by Summer 2016.  To sign up for a better HSV antibody test today, please send an email stating your name, phone number, and a permanent email address to “rvx.abvic.test@gmail.com,” and RVx will contact you once the CLIA certification of our new diagnostic facility is complete.

Below, I explain why the HSV-Type-Specific ABVIC Test is head-and-shoulders better than any other HSV Antibody test currently on the market.

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2.  PRACTICAL Advantage of HSV-Type-specific abvic?

The results I communicate in this section are not theoretical or based on animal studies.  Rather, these are studies performed with actual human patients.  As described to and approved by my university’s institutional review board, my lab has received 45 serum samples over the past year from Westover Heights Clinic in Portland, Oregon in collaboration with Terri Warren.

17 of the serum samples were controls:  ♦ HSV seronegative;  ♦ HSV-1-seropositive;  ♦ HSV-2 seropositive;  or ♦ HSV-1,2-double-seropositive.

The other 28 serum samples were from individuals who had received equivocal results that implied, but did not prove, they were life-long carriers of the HSV-2 virus.  Specifically, each of these patients had been told they might have HSV-2 because they received an Indeterminate diagnosis from antibody tests (often multiple rounds of tests).  Specifically, most patients would first receive the following results:

  1.  A HerpeSelect test that indicated they were low-HSV-2 antibody positive (Index Value of 1 to 5); and everyone in our study would have secondarily ordered a………
  2.  Herpes Western Blot which had yielded Indeterminate results because “ghost bands” appeared at unexpected positions on the blot calling into question if the bands in the HSV-2 lane really represented the presence of HSV-2-specific antibodies or were just more “ghost bands” [i.e., high background).

In the first 34 serum samples sent by the Westover Heights Clinic, my lab had no prior knowledge of which samples were which, and we performed a blinded analysis with our new HSV Type-Specific ABVIC Test.  All of the control serum samples were correctly and immediately diagnosed by the ABVIC Test in 100% agreement with HerpeSelect and HSV Western Blot (i.e., these are the easy samples that all of the tests get right).  In contrast, 16 of the 17 Indeterminate Samples that had confounded both HerpeSelect and HSV Western Blot Tests proved to be HSV-2 seronegative.  Each of these serum samples represents one person who was informed by the Current Standard of Herpes Antibody Testing led that they “might” have HSV-2 genital herpes, and they should live their life accordingly and not risk transmitting the virus (that they did not carry) to others.  However, in these 16 of 17 cases, the quantitatively superior HSV-Type-Specific ABVIC revealed the plain and simple truth that these patients simply had serum that yielded “High Background” on all three of the antibody tests (HerpeSelect, Western, and ABVIC).  Because the ABVIC test is internally controlled, high background is easily distinguished from “low HSV-2-positive.”  To date, of 25 Indeterminate samples screened, only 1 of 25 is actually low HSV-2-positive which means that the vast majority of HerpeSelect tests that yield Index Values in the range of 1 to 4 are most likely false-positives.  With the creation of the new HSV-Type-Specific ABVIC test, there is now a simple test to resolve likely HSV-2-false-positive diagnoses.

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3.  Why is HSV-Type-Specific ABVIC Better?

HerpeSelect and HSV Western blot tests are what is known as qualitative tests, or +/- tests.  Qualitative tests are fine provided that what you are testing for is black or white with no gray are in between.  However, in practice, when one makes scientific measurements (such as measuring the amount of HSV-2-specific antibody in blood), there will be many samples that fall in the gray area between the extremes.  It is here in this gray area that qualitative (+/-) tests are prone to fail because it becomes a subjective guess of the observer (scientist) as to where the dividing line between “negative” and “positive” should be drawn.

In contrast to HerpeSelect and HSV Western Blot, the HSV-Type-Specific ABVIC Test is highly quantitative over a 100-fold dynamic range of measurements and it is the highly quantitative nature of the test that makes it far more robust, objective, and amenable to statistical analysis such that the threshold between “negative” and “positive” may be more reliably drawn.  So what scientific facts back up this claim that the HSV-Type-Specific ABVIC Test is a more quantitatively reliable test?

  1.  Only the ABVIC Test is internally controlled, which allows the test to differentiate “high background” patient serum samples from patient serum that contains “low levels of HSV-2-specific antibodies.”  In the absence of an internal control, it is not surprising that 5% of HerpeSelect tests will mislead patients and/or doctors into believing they have HSV-2 because “high background serum” will return a low-positive index value in the range of 1 to 5.
  2.  Only the ABVIC Test uses a flow cytometer that can sample 1000s of test cells per second.  Thus, each ABVIC Test can compare the efficiency with which a patient’s antibodies bind to (1) uninfected background control cells versus (2) HSV-1+ cells versus (3) HSV-2+ cells.  The preferential binding of a patient’s serum antibodies to thousands of HSV-2+ cells may be compared to thousands of uninfected background controls in 20 seconds such that each “DIAGNOSIS” is based upon tens of thousands of quantitative measurements, which are then subjected to statistical analysis so that a patient may learn the probability that they are infected with HSV-2.  In carpentry, the idiom goes “Measure twice, cut once.”  In arriving at the correct conclusion of whether or not a person possesses blood antibodies to HSV-2, I would suggest that procedurally it makes the most sense to make 1000s of quantitative measurements of the level of patient antibody that binds all 75 proteins in HSV-2-infected cells (full antigenic breadth) versus antibody binding to uninfected cells that serve as an internal background control.  This is what the HSV Type-Specific Test offers, and our forthcoming publication on a study of human subjects suggests that the method can reduce the rate of false-positive HSV-2 diagnoses by at least 90%.   RVx will be offering the HSV-Type-Specific ABVIC Test for patients in need of this service at a cost of $250, and the test can be completed discreetly in less than two weeks.

RVx’s HSV-Type-Specific ABVIC Test (cost $250).  RVx’s website will be live by Summer 2015.  To sign up for a better HSV antibody test today, please send an email stating your name, phone number, and a permanent email address to “rvx.abvic.test@gmail.com,” and RVx will contact you once the CLIA certification of our new diagnostic facility is complete.

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 4.  WHAT DO HSV-Type-Specific Test RESULTS LOOK LIKE?

In a subsequent post, I will explain in detail how the HSV-Type-Specific ABVIC Test works.  For now, I simply describe the type of results a patient would receive and I will try to keep the  technical details to a minimum.

The basis of the test are fixed and permeabilized cells (i.e., stable cells with lots of holes in them so antibodies can access nearly all of the proteins inside the cells).  All of the descriptive words that follow refer to the Figure below of a representative patient’s results.  In total, three different ABVIC tests are run, and these are (1) the ABVIC^HSV test which tests for total antibodies to HSV-1 and/or HSV-2; (2) the ABVIC^HSV-1 test which tests for only HSV-1-specific antibodies; and (3) the ABVIC^HSV-2 test which tests only for HSV-2 -specific antibodies.

Three populations of cells are included in all of the test and these are:

  1.  Uninfected cells that contain no viral proteins.  In the graphs below, these are shown on the left and are labeled “UI cells.
  2. HSV-1-infected cells that contain all of HSV-1’s ~75 proteins.  In the graphs below, these are shown in the middle and are labeled “HSV-1+ cells
  3. HSV-2-infected cells that contain all of HSV-2’s ~75 proteins.  In the graphs below, these are shown on the right and are labeled “HSV-2+ cells

The flow cytometer can distinguish these three populations of cells because of a trick we use in the lab called CFSE-differential labeling.  It does not matter how it is done, but it allows the flow cytometer to segregate the three populations into three separate columns.

Next, a small dilution of the patient’s serum (cell-free fraction of blood) is added to the “Test Cells (UI cells + HSV-1 cells + HSV-2 cells) and the antibodies are allowed to bind any of the 75 viral proteins present in HSV-1+ or HSV-2+ cells.  In addition, at some rate antibodies will non-specifically stick to all of the cells and this is called the “background” of the test.  The background of the test for a given sample is defined by the amount of antibody that sticks to the “UI cells” which serve as an internal background control.

Final steps…….

Excess patient antibodies are rinsed from test cells and a “far-red secondary antibody” that binds human IgG antibody (potentially stuck to HSV-2 proteins) is added to the test cells.  The more HSV-1 or HSV-2-specific antibody present in a patient’s serum sample, the more human antibodies are stuck to the HSV-1+ cells and/or HSV-2+ cells and the more of the “far-red secondary antibody” gets stuck to those same cells.  At the end of this second labeling step, excess secondary “far-red” antibody is washed from the test cells.

KEY CONCEPT DEFINED HERE:  The more “HSV-specific antibody a person has in their serum,” the more far-red color will preferentially stick to the HSV-1+ or HSV-2+ cells compared to UI cells, and thus the higher those cell populations will “shift up” the y-axis……..higher on the y-axis equals brighter red = more serum antibody stuck to a sub-population of test cells.

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TEST RESULTS EXPLAINED FOR JOHN A. DOE

In the first ABVIC^HSV Test at the top of the Figure, serum from John A. Doe was combined with the Test cells and revealed that this person is “HSV-seropositive” because both HSV-1+ cells and HSV-2+ cells are shifting upwards.  This first test does not distinguish whether this person has HSV-1 or HSV-2, but the test results indicate that the probability of this person being HSV-seronegative (not infected with either virus) is less than one in a million.Sample HSV Type-Specific ABVIC

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In the second ABVIC^HSV-1 Test in the middle of the Figure, serum from John A. Doe was first pre-absorbed to HSV-2+ cells to remove all (1) type-common antibodies shown as blue ‘Y’s in the top right box and to remove and (2) HSV-2-specific antibodies shown as green ‘Y’s in the top right box.  Hence, the only population of HSV-specific antibodies that could be left would be the red ‘Y’s that are HSV-1-specific antibodies.  In the case of John A. Doe, the individual has no HSV-1-specific antibodies as shown in the middle graph because the HSV-1+ test cells are no “redder’ (shifted up the y-axis) than the HSV-2+ test cells.  Based on statistical analysis, John A. Doe is HSV-1-seronegative.

In the third ABVIC^HSV-2 Test at the bottom of the Figure, serum from John A. Doe was first pre-absorbed to HSV-1+ cells to remove all (1) type-common antibodies shown as blue ‘Y’s in the top right box and to remove and (2) HSV-1-specific antibodies shown as red ‘Y’s in the top right box.  Hence, the only population of HSV-specific antibodies that could be left would be the green ‘Y’s that are HSV-2-specific antibodies.  In the case of John A. Doe, the individual has HSV-2-specific antibodies as shown in the bottom graph because the HSV-2+ test cells are “redder’ (shifted up the y-axis) than the HSV-1+ test cells.  Based on statistical analysis, John A. Doe has less than a one-in-a-million probability of being HSV-2-seronegative.  Hence, John Doe’s blood test offers definitive proof that he is infected with the HSV-2 virus.

In subsequent posts, I will consider the HSV-Type-Specific ABVIC Test in greater depth and examine the underlying reason that it is 100 times more sensitive than the current standard of care in herpes antibody testing around the world; namely, Focus Diagnostics’ HerpeSelect Test.

– Bill H.

12 thoughts on “HSV ABVIC Test

  1. TVEC says:

    Bill,

    It is very interesting and exciting that you have been able to show that the equivocal result is almost certainly a negative result.

    I think it would also be very interesting to know what the false negative rate of HerpeSelect and WB are as resolved by the ABVIC. There seems to be a lot of speculation that the HerpeSelect sensitivity is not as high as advertised… I believe the quoted figure for HerpeSelect is 97% sensitivity to HSV2 after 12-16 weeks post-infection (HSV1 sensitivity is worse than this). However here is one study that indicated that the false negative rate is higher:

    http://sti.bmj.com/content/early/2016/01/11/sextrans-2015-052213.abstract

    The study doesn’t use the term “HerpeSelect” but I’m guessing that the tests they used are very similar to HerpeSelect. They claim a false negative rate of ~15% for HSV2, which seems quite dramatic. Terri Warren on the other hand seems to be confident in the published sensitivity of the HerpeSelect test, presumably because it is consistent with what she sees in her clinic.

    The ABVIC test did confirm all the serum samples that were not in the equivocal range, so I guess that bodes well for the HerpeSelect and WB (small control sample size, I know)

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    • Herpes Vaccine Research says:

      Hi TVEC,

      In practical terms, HerpeSelect is a lame idea for the same reasons that gD subunit vaccines are a lame idea. HerpeSelect is based on antibodies against a single HSV-1 or HSV-2 protein….glycoprotein G. There are two problems with this.

      The first problem is that the antibody response to HSV-1 or HSV-2 is directed against ~20 viral proteins, and so one big problem is that 95% of the relevant viral proteins are missing from the HerpeSelect test…..hence rendering it incredibly prone to yielding false-negatives during the first 3 to 6 months of a HSV-1 or HSV-2 infection. It should only take 2 to 3 weeks for newly HSV-infected persons to “seroconvert” if the antibody test being used included ALL of HSV-1 or HSV-2’s antigens, and was not based solely on antibodies to glycoprotein G of HSV-1 (gG-1) or glycoprotein G of HSV-2 (gG-2).

      A second problem emerges when one actually, as crazy as this is, takes the time to study and understand the antibody response to HSV and that problem can be summarized in a single word………immunodominance. If one takes all of HSV-1 or HSV-2’s proteins and rank orders them in terms of “What fraction of the total antibody response to HSV” is directed against a single protein, one may develop a list called an immunodominance heirarchy. I have done this ad nauseum with serum from (1) mice vaccinated with a live HSV-2 vaccine; (2) guinea pigs vaccinated with a live HSV-2 vaccine; and (3) human beings who have been naturally infected with HSV-2. Based on my studies, the top 5 most dominant HSV-2 antigens include proteins like UL39 (RR-1), ICP8, VP1-2, VP5, and glycoprotein B. Please note that gG-2 (the basis of HerpeSelect) is not included in the list of HSV-2’s top five antigens. In fact, in my studies, glycoprotein g of HSV-2 (gG-2) is about the 20th most dominant HSV-2 antigen, which meams that I believe it is an absolutely trivial HSV antigen.

      In scientific terms, the HerpeSelect test is used to differentiate whether people have HSV-2, or not, based on the presence of relatively rare HSV-2 antibodies…perhaps 0.5% of the total population of “HSV-2 specific antibodies.” This is poor science to develop a diagnostic test that relies on such a minor fraction of the “HSV-2-specific antibodies” we are trying to detect. In contrast, the ABVIC test analyzes the presence of 100% of the HSV-2-specific antibodies, which explains why it is at least 100 times more sensitive than HerpeSelect in head-to-head tests.

      In practical terms, HerpeSelect is a bad idea……..a crappy test with an unacceptably high rate of false-positives (about 10% by my estimates) and an unacceptably slow serocoversion time…..3 months instead of the ~2 weeks it should take. This is very typical of how herpes scientists have operated for the past 30 years….let’s offer a lame solution in the 1980s and then become complacent and not evaluate whether or not the initial “stopgap solution” was all that or whether there were better options we could develop.

      Let me be unequivocal on this point……..there are at least three completely different reasons that the HerpeSelect test is a spectacularly inferior test. Yet one more piece of evidence that suggests a pattern of apathy and laziness in my field when it comes to the fact that herpes virologists / immunologists dedicate far to little of their training and brain power to thinking about how best to help their fellow man and reduce the human suffering caused by herpes simplex virus. In the past 20 years, the sciences of immunology and virology have radically advanced but virtually none of that new knowledge has been harnessed, distilled, and used to offer patients and doctors better solutions to deal with the proper diagnosis and prevention of herpetic disease. I find this complacency towards human suffering to be both unacceptable and highly offensive. Yes, science geeks are prone to retreat to their ivory towers. In the herpes field, the problem is that they never emerge from the ivory towers long enough to talk to the millions of people who actually suffer with the disease they are supposedly “trying to solve.”

      This is why I formed RVx, and the ABVIC test will (finally) solve the age-old problem of how to precisely and accurately diagnose HSV-1 and HSV-2 infections. In practical terms, ABVIC vs HerpeSelect is like an automobile race between a Ferrari versus a Model T…..there is simply no comparison.

      With the launch of the ABVIC Test, I anticipate it will become very clear over the next 1 to 2 years how much radically better herpes serology testing can be when one uses the flow-cytometry-based ABVIC test, which is a statistically-based quantitative test based on n=10,000 to 30,000 measurements per patient sample. This is in comparison to the n=1 to 2 measurements that HerpeSelect uses to decide whether a patient is “HSV-2 seronegative” or “HSV-2 seropositive.” Gotta get the website up and publish our results, and then it will be easier for you to see exactly what I mean.

      – Bill H.

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  2. Branwell says:

    Hello,

    For the test that is scheduled in summer 2016, would taking a suppressive dosage of valacyclovir (500mg/day) confound it? If so, could you offer some timing guidance on how long one must not take V before the blood draw for the test, so that it won’t confound the test at all?

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  3. DQ2016 says:

    I think it is quite genius that you came up with a better testing test for HSV! I’m going to purchase a kit once available and it will help me contribute to your/our campaign against HSV. I wouldn’t hesitate to buy one and it feels good to actually get something back in return which is a solid deal. I’m sure many others feel the same way as I do. Once again, I appreciate your hard work, passion and dedication and feel a little hope with your vaccine possibly on it’s way.

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    • G says:

      Second this, will buy a kit when made available. A part of me hopes some type of vaccine is made available along with the kit. It’s depressing realizing you have this and are limited to ineffective options (Valtrex) to help make it better. It would be comforting to receive positive results, then given suggested dates in which one could receive a vaccine in the future if wanted. Professor Halford we hope you and RvX are able to make all your offerings available for your desperate and needy supporters.

      Thank you for taking time to explain and respond to all questions on this virus.

      Best,
      G

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  4. SilverFox says:

    Hi Bill,

    I believe my past testing history may be of interest to some of your readers.

    Nov 2011 – swabbed positive for hsv1

    Dec 2011 – IGG type specific tested neg for hsv1 low pos for type 2 at 2.10

    Mar 2012 – IGG type specific tested neg for hsv1 low pos for type 2 at 1.10

    April 2012 Western Blot tested neg for hsv2 indeterminate for hsv1. It was suggested that I test again in 6 months so that they could do a convalescent study. Basically, comparing my first WB with a second WB.

    April 2016 Just took another WB through Terri Warren’s clinical trial. Results came back Neg for hsv1 and indeterminate for hsv2. This is ridiculous! WB is supposed to be the gold standard for testing and my two WB’s are totally opposite.

    My testing history is a joke. Is it really too much to ask to know if I actually have hsv and which type???

    Sorry to sound skeptical, but if I chose to spend another $250 to take your test, will I finally know?

    Thanks,
    SF

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    • Herpes Vaccine Research says:

      Hi SF,

      Yes, RVx’s ABVIC test will give you a definitive answer because it is (1) highly quantitative, (2) based on 1000s of replicate measurements per test (not n=1 like HerpeSelect or Western Blot), and because it is (3) internally controlled. The ABVIC test compares how your serum IgG antibodies bind to uninfected cells, HSV-1-infected cells, and HSV-2+ cells. Your serum (blood) likely produces high background and that is why you keep getting “low positive” results…..because your background is higher than normal. The ABVIC test can tell the difference because it does not matter how much “IgG antibody binds to uninfected cells” (background). What matters is that the ABVIC test (a flow cytometry-based test) compares the quantity of antibody binding to (1) 1000s of uninfected cells versus (2) 1000s of HSV-infected cells. With those types of numbers per measurement, it is much harder to make mistakes with the internally controlled and highly quantitative ABVIC test. In contrast, the far lamer (more qualitative) HerpeSelect and Western Blot tests cannot differentiate (1) serum with higher than average background from (2) serum that contains low levels of HSV-specific antibody. The ABVIC (antibody-binding to virus-infected cells) test should be online and available by August 2016. I will announce on the blog when RVx’s forthcoming website goes live, and the test will be available the day the website goes live.

      – BH

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      • SilverFox says:

        Bill,

        Thank you for the information and explanation. I have been told that the most accurate of all is a blister that has been swabbed. I had one small blister initially in Nov 2011 and was told it swabbed pos for hvs1. I have never had another blister or sign that I have hsv apart from the tests results I posted above. What confuses me is that the 2 igg’s both show neg for hsv1 and out of 2 WB’s, 1 was indeterminate for hsv1 and the other negative.

        Are swabs really the most accurate? Or is your new test more accurate?

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      • Herpes Vaccine Research says:

        Hi Silverfox,

        Swabs are definitive when they come back virus-positive. However, they have a false-negative rate of at least 50% so a negative result does not mean that you do not have HSV-1 or HSV-2. Yes, the ABVIC test would be far more reliable.

        – Bill H.

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      • SilverFox says:

        I think your test is going to be very important based on the explanation you have given. It has become painfully clear to me that the current testing options are inaccurate and unreliable, as I have displayed in my conflicting results in an earlier post. Terri Warren reached back out to me and said she spoke with the team at the University of Washington about my results. The bottom line is that neither the two IGG’s or 2 WB’s gave accurate feedback to my condition. At the end of the day they had to refer back to the positive swab, and concluded that I have hsv1 gen, and not hsv2.

        If my testing would have been accurate, I would not have had to take multiple tests, been filled with frustration, nor spent close to $1,000 in testing.

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      • Herpes Vaccine Research says:

        Hi SilverFox,

        I believe that you have very succinctly and eloquently stated precisely my position, but in a way that will be very relatable for individuals who have previously received equivocal herpes test results. Yes, my company is offering the ABVIC test for precisely the reasons you highlight…..the current qualitative tests work most of the time for most people, but fail 10% of the time which is an unacceptably high failure rate when what hangs in the balance is whether a doctor is going to say “I think you have genital herpes but your test result does not definitively prove my belief.” This sucks, and like many things in my field, is an unacceptably bad solution that has been adopted out of laziness and apathy….not because it is the best course of action. I fully intend for my company (RVx) to solve ALL of the herpes problems including (1) rampant misinformation and (2) the commonplace use of inferior herpes tests that are inadequate for the magnitude and gravity of the job of definitively diagnosing people with HSV-1 and/or HSV-2 infections.

        – Bill H.

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